Rapid Identification of
            <i>Staphylococcus aureus</i>
            Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

Oliveira, Kenneth; Procop, Gary W.; Wilson, Deborah A.; Coull, James; Stender, Henrik

doi:10.1128/jcm.40.1.247-251.2002

Cited by 165 publications

(116 citation statements)

References 21 publications

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“…The previously published FISH assays were implemented as described previously (7,12,17,18,27) (Table 2). New DNA probes were designed for N. meningitidis and E. coli using the ARB program package, freely available at http://www.arb-home.de/.…”

Section: Bacterialmentioning

confidence: 99%

“…Examples are the detection and identification of pathogens in blood cultures, tissues, and cell cultures (2,7,12,17,19,27). FISH is a cheap and quick method that does not require costly technical equipment.…”

mentioning

confidence: 99%

“…Its utility for diagnosis of bacterial meningitis has, however, not been determined yet. Therefore, we defined a FISH probe set for the detection of bacterial pathogens in cerebrospinal fluid that includes previously published probes (2,7,12,17,27) as well as newly designed probes for the detection of N. meningitidis, E. coli, and Staphylococcus spp.…”

mentioning

confidence: 99%

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Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

et al. 2005

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Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy-and culture-positive samples (n ‫؍‬ 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy-and culture-negative samples (n ‫؍‬ 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.

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Section: Bacterialmentioning

confidence: 99%

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confidence: 99%

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Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

et al. 2005

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“…Additionally PCR is unable to distinguish dead bacteria from live cells, and the required reagents are relatively costly. Gene probe assays are promising in that they offer simple, rapid, and sensitive measurements and are lower in cost than PCR techniques (2,6). Several rapid methods (culture-based and molecular-based screening methods) for S. aureus are also available, allowing diagnostics within hours of collection time; however these tests can be costly and the majority yield qualitative, not quantitative results (8 -10).…”

mentioning

confidence: 99%

“…Traditional identification through plate-culture requires 2-3 days, can require subculturing or biochemical analysis (6), and necessitates blood sample volumes difficult to obtain in pediatric patients (7). Molecular methods, such as polymerase chain reaction, reverse transcription-polymerase chain reaction, and quantitative reverse transcription-polymerase chain reaction (PCR, RT-PCR, and qRT-PCR) are primarily based on particular genes specific to S. aureus (2) and offer the most sensitive measurements in the least amount of time, with the least amount of sample, but can be prone to ambiguous results that can only be resolved through sample cultivation (5).…”

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confidence: 99%

Viable Staphylococcus aureus Quantitation using 15N Metabolically Labeled Bacteriophage Amplification Coupled with a Multiple Reaction Monitoring Proteomic Workflow

Pierce

Rees

Fernández

et al. 2012

Molecular & Cellular Proteomics

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A multiple reaction monitoring liquid chromatography method with tandem mass spectrometric detection for quantitation of Staphylococcus aureus via phage amplification detection is described. This phage amplification detection method enables rapid and accurate quantitation of viable S. aureus by detecting an amplified capsid protein from a specific phage. A known amount of metabolically labeled 15 N reference bacteriophage, utilized as the input phage and as the internal standard for quantitation, was spiked into S. aureus samples. Following a 2-h incubation, the sample was subjected to a 3-min rapid trypsin digest and analyzed by high-throughput liquid chromatography tandem mass spectrometric detection targeting peptides unique to both the 15 N (input phage) and 14 N (progeny phage) capsid proteins. Quantitation was achieved by comparing peak areas of target peptides from the metabolically labeled 15 N bacteriophage peptide internal standard with that of the wild-type 14 N peptides that were produced by phage amplification and subsequent digestion when the host bacteria was present. This approach is based on the fact that a labeled species differs from the unlabeled one in terms of its mass but exhibits almost identical chemical properties such as ion yields and retention times. A 6-point calibration curve for S. aureus concentration was constructed with standards ranging from 5.0 ؋ 10 4 colony forming units (CFU) ml ؊1 to 2.0 ؋ 10 6 CFU ml ؊1 , with the 15 N reference phage spiked at a concentration of 1.0 ؋ 10 9 plaque forming units (PFU) ml ؊1 . Amplification with 15 N bacteriophage coupled with LC-MS/MS detection offers speed (3 h total analysis time), sensitivity (LOD: < 5.0 ؋ 10 4 CFU ml ؊1

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Labeling of Bacterial Pathogens for Flow Cytometric Detection and Enumeration

Harkins¹,

Harrigan²

2004

CP Cytometry

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Traditional uses of flow cytometry have been for research and diagnostic purposes, on mammalian cells. This unit focuses on using a flow cytometer for enumerating specific bacteria and parasites. Several different labeling methods are discussed, as well as how to set up a cytometer for detecting and enumerating bacteria. Labeling methods include direct detection with a primary fluorochrome‐conjugated antibody and indirect labeling with an unconjugated primary and a fluorochrome‐conjugated secondary antibody, as well as labeling with rRNA sequence‐specific peptide nucleic‐acid probes end‐labeled with a fluorochrome. Data and methods throughout this unit focus on detection of Salmonella in clean matrices; however, pertinent information is also provided on using these methods to label other pathogens. The Commentary covers critical aspects of the protocols, and also includes information and suggestions on the application of these methods for testing in the food and pharmaceutical industries, as well as in environmental water testing.

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Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

Cited by 165 publications

References 21 publications

Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

Viable Staphylococcus aureus Quantitation using 15N Metabolically Labeled Bacteriophage Amplification Coupled with a Multiple Reaction Monitoring Proteomic Workflow

Labeling of Bacterial Pathogens for Flow Cytometric Detection and Enumeration

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